281 lec29 mol_tech1

Lecture 29:

Molecular techniques I. DNA extraction

Readings (chapter 10)

Course 281

Lessons for life

AIMS

• Understand the sources of DNA in different organisms.

• Understand the cellular structure that needs to be disrupted to extract DNA.

• Understand the process of DNA isolation and the different methods employed.

• Understand the reasons for using specific chemicals in DNA extraction and what each does.

Cell Structure

Cells and DNA

• In an animal cell, DNA is found in the nucleus and mitochondria.

• In a plant cell, DNA is found in the nucleus, mitochondria, and chloroplast.

• In bacteria, DNA is found in plasmids and bacterial chromosome.

Where Do we get the DNA from?

Cells and DNA

• The mitochondria and chloroplast resembles a bacteria in genome structure.

• Knowing the DNA source you are targeting is essential.

Cells and DNA

In humans, where can we get DNA from?

Cell Structure

• All living cells contain hereditary information coded in DNA (some exceptions! What are they?).

• DNA in human cells is found in the nucleus and mitochondria.

• The DNA of the mitochondria resembles that of bacteria.

• The DNA in the nucleus is packaged into 22 pairs of autosomal chromosomes and one pair of sex chromosomes (XX or XY – some exceptions).

Human DNA sources

Blood is a connective tissue that is rich in proteins

Plasma

No DNA

Erythrocytes (RBC)

• No nucleus ➔ No DNA! • Birds and reptiles?

Leukocytes (WBC)

• Nucleus ➔ DNA!

Human DNA sources

Mammals RBC Birds RBC

Human DNA sources

• We can get DNA only from living cells.

• Can we get DNA from trimmed hair?

• Can we get DNA from teeth enamels?

• What about bone?

Human DNA sources

Human DNA sources

• Easy to collect and DNA is easy to extract.

• Saliva samples contain cells (mostly epithelial) and can be a good source of DNA.

• Check 23andme.com

Human DNA sources

Cells and DNA

What about other organisms?

Bacterial DNAWe can grow bacteria in the lab and extract

chromosomal DNA and plasmid DNA.

plate Flask

Plant DNA

Plants contain living cells and non-living cells

Plant leaves are good source of DNA. Other tissue may be hard to get DNA from.

Animal DNAAnimals are not different from humans!

Not every animal tissue has DNA!

Getting DNA

How to get DNA out of the cell?

Getting DNA

A new way to get DNA ☺

DNA isolation

1. Cell and tissue disruption 2. Lysis of cell membrane 3. Separating DNA from the rest of the

cell content.

DNA isolation1. Cell and tissue disruption

Enzymatic (proteinase K)

Grinding (liquid N)

Boiling (Alkali)

DNA isolation2. Lysis of cell membrane

What is the cell membrane? What does the cell membrane contain?

Cell membrane

Cell membrane

Phospholipid molecule

• Hydrophilic ? • Hydrophobic?

DNA isolation2. Lysis of cell membrane

• Cell membrane contains:

• Phospholipid bilayer. • Proteins. • Other molecules.

• We need to open the cell membrane through cell lysis.

• We need to remove the proteins and other molecules.

DNA isolation

Lysis buffer: • Detergent (SDS) • Buffer (Tris-HCl) • Salt • Reducing agent

(mercaptoethanol) • Chelating agent (EDTA)

2. Lysis of cell membrane

DNA isolation

DNA isolation3. Separating DNA from the rest

Organic extraction (phenol-chloroform extraction)

Column extraction (DNA selective binding)

Phenol-chloroform extraction protocol

1: 0.2-0.25g of dissected tissues in 2ml

tubes

or

2: Chill the mortar and

pestle with liquid N2

3: Place tissue in the mortar and pestle and pour liquid N2

4: Grind tissue until

obtaining fine powder

Note: keep pouring

liquid N2

6: Add 3ml of digestion buffer to the powdered samples

5: Transfer powdered samples into 15ml tubes

7: Incubate @ 37º C +

rotate tubes

Incubation period 1 hr

8: Add 3ml of P.C.I.

+ mix by inverting

for 4 min + 1 min to

settle down

9: Transfer aqueous layer into anew

15ml tube

10: Repeat twice

11: Add 3ml of C.I.

+ mix by inverting

for 4 min + 1 min to

settle down

12: Transfer

aqueous layer into

anew 15ml tube

13:

Rep

eat

on

ce

13: Pipette 500ul of the

aqueous layer into 3x 2ml

tubes

14: Add 1000ul of ice cold

ethanol and 150ul of sodium

acetate into each tube

15: Mix by inverting +

Incubate @ -20 º C for 1hr

16: Thaw tubes + centrifuge

@ 13000 rpm for 30 min

START RNA ISOLATION17: collect pellet -

tilt tube + pour the

solution

18: dissolve pellet

in 1ml 70% ethanol

• 19: Centrifuge @

13000 rpm for 15 min

• 20: Collect pellet as

described in 16

• 21: Dry pellet for 30 min

in a vacuum dessicator

• 22: Resuspend pellet with

500ul ddH2O + incubate in

37º C water bath overnight

• 23: Store in in -20º C

• START DNA EXTRAXTION

BY A COMMERCIAL KIT

Phenol-chloroform extraction protocol

1: Mix whole blood then pipette 350ul

2 ml tube

2: Add 500ul Lysis

solution A

Invert to mix 3: Incubate @ 65°C for

6 min + invert to mix

4: Add 900ul chloroform

Vortex 5: Add 200ul

precipitation solution

B + Vortex

6: Centrifuge @ max

speed for 5-10 min

7: Transfer aqueous layer

into 2ml tube

8: Add 1ml of 100%

ethanol

Invert to mix

Until a precipitate

appears

8: Centrifuge @ max

speed for 5 min

9: Collect pellet - tilt tube

+ pour solution

10: Add 1ml of

70% ethanol 11: Centrifuge @

max speed for 2 min12: Pour off the ethanol

13: Centrifuge @

max speed for 1 min

14: Dry the pellet in

the oven for 5 min

15: Resuspend pellet

with 150ul ddH2O

Incubate @ 65°C for 5 min

16: Label tube + store

@ -20 °C

Column extraction protocol

How it looks?

The secret of life in a speck of jelly! (Mendel’s dwarf)

DNA storage

• DNA is stored in TE buffer to ensure stability and inhibit DNase.

• Long term storage is done using -80 freezers.

• -4 and -20 freezers are used for frequent use of the DNA sample.

• Freezing and thawing a sample may cause damage to DNA.

To know

Genomic DNA

Mitochondrial DNA

Plasmid DNA

Chloroplast DNA

RBC in mammals

RBC in birds

WBC DNA

Cell and tissue disruption

Cell lysis

DNA isolation

Cell membrane

hydrophilic

Hydrophobic

Detergent

salt

Reducing agent

Organic extraction

Column extraction

Phospholipid bilayer

DNA storage

Expectations

• You know the sources of DNA in various organisms.

• You know not all material from a living organism can be a source of DNA.

• You know the process of isolating DNA.

• You know the chemicals and what they do in DNA extraction protocol.

• You know the two major protocols of DNA extraction.

• You know DNA storage methods and stability.

For a smile